3D multicolor super-resolution imaging offers improved accuracy in neuron tracing.
AuthorsLakadamyali M, Babcock H, Bates M, Zhuang X, Lichtman J,
JournalPLoS One
PubMed ID22292051
'The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic ... More
Preparation of photoswitchable labeled antibodies for STORM imaging.
AuthorsBates M, Jones SA, Zhuang X,
Journal
PubMed ID23734027
'Stochastic optical reconstruction microscopy (STORM) is a method for superresolution fluorescence imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. Many synthetic fluorescent dye molecules show photoswitchable fluorescence ... More
CD8 T cell competition for dendritic cells in vivo is an early event in activation.
AuthorsWillis RA, Kappler JW, Marrack PC
JournalProc Natl Acad Sci U S A
PubMed ID16880405
'T cell responses against an antigen are often focused on a small fraction of potentially immunogenic determinants, a phenomenon known as immunodominance. Immunodominance can be established at several stages of antigen presentation, including antigen processing, binding of peptides to MHC, and competition between T cells for dendritic cells (DCs). The ... More
Fast, three-dimensional super-resolution imaging of live cells.
AuthorsJones SA, Shim SH, He J, Zhuang X,
JournalNat Methods
PubMed ID21552254
'We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model ... More
Superresolution imaging of chemical synapses in the brain.
AuthorsDani A, Huang B, Bergan J, Dulac C, Zhuang X,
JournalNeuron
PubMed ID21144999
'Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here, we present a superresolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction ... More
Color-blind fluorescence detection for four-color DNA sequencing.
AuthorsLewis EK, Haaland WC, Nguyen F, Heller DA, Allen MJ, MacGregor RR, Berger CS, Willingham B, Burns LA, Scott GB, Kittrell C, Johnson BR, Curl RF, Metzker ML
JournalProc Natl Acad Sci U S A
PubMed ID15800037
'We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission ... More
Statistical deconvolution for superresolution fluorescence microscopy.
AuthorsMukamel EA, Babcock H, Zhuang X,
JournalBiophys J
PubMed ID22677393
'Superresolution microscopy techniques based on the sequential activation of fluorophores can achieve image resolution of ~10 nm but require a sparse distribution of simultaneously activated fluorophores in the field of view. Image analysis procedures for this approach typically discard data from crowded molecules with overlapping images, wasting valuable image information that ... More
Multicolor super-resolution imaging with photo-switchable fluorescent probes.
AuthorsBates M, Huang B, Dempsey GT, Zhuang X,
JournalScience
PubMed ID17702910
Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable ... More
Molecular architecture and assembly principles of Vibrio cholerae biofilms.
In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels ... More
The protein tyrosine phosphatase SHP-1 modulates the suppressive activity of regulatory T cells.
AuthorsIype T, Sankarshanan M, Mauldin IS, Mullins DW, Lorenz U,
JournalJ Immunol
PubMed ID20952680
The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src ... More
Rac1 activation in podocytes induces rapid foot process effacement and proteinuria.
AuthorsYu H, Suleiman H, Kim AH, Miner JH, Dani A, Shaw AS, Akilesh S,
Journal
PubMed ID24061480
The kidney's vital filtration function depends on the structural integrity of the glomerulus, the proximal portion of the nephron. Within the glomerulus, the architecturally complex podocyte forms the final cellular barrier to filtration. Injury to the podocyte results in a morphological change called foot process effacement, which is a ubiquitous ... More
The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway.
AuthorsTan D, Cai Y, Wang J, Zhang J, Menon S, Chou HT, Ferro-Novick S, Reinisch KM, Walz T,
Journal
PubMed ID24218626
The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals ... More
Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons.
AuthorsXu K, Zhong G, Zhuang X,
JournalScience
PubMed ID23239625
Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced ... More
Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins.
Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements ... More
Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.
AuthorsSmith K, Garman L, Wrammert J, Zheng NY, Capra JD, Ahmed R, Wilson PC,
JournalNat Protoc
PubMed ID19247287
We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, ... More
Rab conversion as a mechanism of progression from early to late endosomes.
AuthorsRink J, Ghigo E, Kalaidzidis Y, Zerial M
JournalCell
PubMed ID16143105
The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo ... More